The present invention relates to new methods for immobilizing biomolecules such as, for example, different proteins, enzymes, monoclonal and polyclonal antibodies, cells, cell organelles, and tissue samples as well as affinity ligands, to a plurality of different polymer carriers as well as methods for manufacturing new affinity ligands reacting with amino groups. Affinity ligands in this context are to be understood to include, in the broadest sense, amino group containing biochemical probes, physiological indicators, antibiotics, enzyme inhibitors, chelates, lipids, tracers, oligopeptides, oligonucleotides, oligosaccharides, respectively, the corresponding monomer components. The chemical bond between the carriers and the ligands are amide-like bonds with a surprisingly high stability. A prerequisite for this coupling is that the polymer carrier as well as the ligand contain primary or secondary aliphatic amino groups, respectively, aromatic amino groups. A further advantage, in addition to the high stability of the bond, with respect to conventional immobilizing methods is the very gentle coupling chemistry which surprisingly allows to use, for example, only peripheral amino groups of proteins in a directed manner for the coupling. In comparison to previously known techniques the inventively used new immobilization results also in a substantial stabilization of many immobilized macromolecules such as immunoglobulins or enzymes so that they are partially protected against denaturation. The chemical xe2x80x9clinksxe2x80x9d connecting to amino groups are derivatives of cyclobutene carboxylic acid (Formula I). Some derivatives of this acid are known (A. H. Schmidt, Synthesis, 1980, 961, K. H. Glusenkamp et al., Z. Naturforsch. C 1991, 46, 498, L. F. Tietze et al., Chem. Ber. 1991, 124, 1215).
Entirely new, however, is the use of this reagent for immobilizing amino group, containing molecules to polymer matrices, which must also contain an amino group, whereby the substituents R1, R2, A, B, PL, and PM of the general formula (I) have the meaning indicated in the description. Either PL and/or PM must be provided in dissolved form in order for immobilization to occur. A special advantage is that, for example, cyclobutene carboxylic acid diethyl esters can be reacted in a two-step reaction with amines to diamides. It is possible in this context to react first a polymer matrix (method A) or a ligand (method B) in a first step with the cyclobutene carboxylic acid diester. In both cases, in the second step a stable amide-analog bond is formed. It must be underscored that the reaction of the cyclobutene carboxylic acid diester with an amine to a monoamid is substantially faster than the subsequent reaction to the diamide. Furthermore, the cyclobutene carboxylic acid diester, cyclobutene carboxylic acid monoamide, and cyclobutene carboxylic acid diamide exhibit characteristic differences in their UV spectra. While cyclobutene carboxylic acid diester has a maximum at 250 nm, the monoester amide has a maximum at 272 nm, respectively, the diamides have a maximum at 290 to 300 nm with very high extinction coefficients. It is thus possible to monitor the degree of immobilization by UV spectroscopy, for example, due to the reduction of an activated ligand (cyclobutene carboxylic acid monoamide) in the reaction solution. Two surprising properties of the cyclobutene carboxylic acid derivatives must be underscored: 1. It has been shown that the cyclobutene carboxylic acid amide bond cannot be cleaved by conventional protease enzymes. This has important consequences for immobilized affinity ligands which are used for applications in biological systems because a substantially higher stability, respectively, life expectancy for this kind of covalent coupling in comparison to conventional amid or ester bonds is to be expected. 2. The surprisingly very selective coupling chemistry opens an unexpected, extremely broad range of reactions. For example, oligonucleotides with primary or secondary amines of any sequence and length can be reacted with cyclobutene carboxylic acid esters in a chemoselective manner without chemically changing the amino groups of the DNA bases. The thus produced oligonucleotides, activated with cyclobutene carboxylic acid groups, can subsequently be immobilized in a controlled manner on polymer matrices, for example, on membranes or latex surfaces. Furthermore, it is, for example, possible to selectively react the reactive oligonucleotides with marker molecules or probes such as, for example, digitoxigeninamine or dyes such as fluoresceine. The only requirement is that the marking molecule or probe is functionalized with amino groups. The surprisingly chemoselective reaction behavior of the cyclobutene carboxylic acid derivatives allows for the manufacture of novel compounds, which, for example, can may become important for the construction of chemosensors and biosensors. Surprisingly, it is, for example, possible to couple functionalized azamacrocycles or chelate forming compounds such as, for example, desferrioxamine B or also aminocyclodextrin with biological or synthetic membranes in a directed manner. It is also possible to activate, for example, lipids such as phosphatidyl ethanolamine derivites with cyclobutene carboxylic acid in order to attach in a second step any suitable marker molecule as, for example, chelates, dyes or also aminocyclodextrins, resulting in new lipids that can then, for example, be integrated in liposomes, can lead to lipisomes with favorable transport properties for active reagent or can lead to liposomes with a selective membrane transmissivity (channels). It is also possible, after activation of a matrix with cyclobutene carboxylic acid groups, to react any polyamine with the activated matrix. The thus directedly introduced further amino groups can then subsequently be again activated with cyclobutene carboxylic acid groups which are then, in turn, reacted with amines. The controlled chemical reaction at polymer surfaces (controllable in a directed manner by excess amounts of reagents) and the unique, stepped reaction behavior of the cyclobutene carboxylic acid diesters, respectively, cyclobutene carboxylic ester amides allows in this manner to generate novel functionalized polymers with comb, rotaxan or dendrimer structure. It is possible to generate extremely active surfaces with a high density of functional end groups which affect the physical and chemical properties in a considerable manner and, for example, are also able to stabilize very large molecules or even cells with a multitude of covalent or non-covalent bonds in a selective manner. On the other hand, it is possible in the same manner to bond in a directed manner low molecular compounds, after activation with cyclobutene carboxylic acid groups, with functionalized polymers.
The inventive method for manufacturing immobilized biomolecules and affinity ligands is primarily characterized by the steps of:
a) selecting a compound from amino group containing biomolecules and amino group containing affinity ligands and dissolving the selected compound;
b) reacting the selected and dissolved compound of step a) with a cyclobutene carboxylic acid derivative, selected from the group consisting of cyclobutene carboxylic acid diester, cyclobutene carboxylic acid halide, cyclobutene carboxylic acid dialkoxyester, cyclobutene carboxylic acid imidazole, in an inert solvent to form a cyclobutene carboxylic acid adduct;
c) dissolving a compound containing amino groups;
d) reacting the cyclobutene carboxylic acid adduct with the dissolved compound containing amino groups of step c) at a pH of 7-10 and a temperature of +4xc2x0C. to +60xc2x0C. in an aqueous buffer system, free of primary and secondary amines, to form an intermediate product with covalent cyclobutene carboxylic acid bridges; and
e) reacting the intermediate product with covalent cyclobutene carboxylic acid bridges with a low molecular weight compound having active amino groups in an aqueous buffer system, free of primary and secondary amines, at a pH of 7-10.
The selected compound preferably contains at least one amino group selected from a primary amino group and a secondary amino group.
The selected compound is preferably a polymer.
The polymer is expediently a matrix.
The low-molecular weight compound preferably contains at least one amino group selected from a primary amino group and a secondary amino group.
The invention also relates to a method for manufacturing immobilized biomolecules and affinity ligands characterized by the following steps:
a) selecting a compound from amino group containing biomolecules and amino group containing affinity ligands and dissolving the selected compound;
b) reacting the selected and dissolved compound of step a) with a cyclobutene carboxylic acid derivative, selected from the group consisting of cyclobutene carboxylic acid diester, cyclobutene carboxylic acid halide, cyclobutene carboxylic acid dialkoxyester, cyclobutene carboxylic acid imidazole, in an inert solvent to form a cyclobutene carboxylic acid adduct;
c) dissolving a compound containing amino groups;
d) reacting the cyclobutene carboxylic acid adduct with the dissolved compound containing amino groups of step c) in an inert solvent to form an intermediate product with covalent cyclobutene carboxylic acid bridges; and
e) reacting the intermediate product with covalent cyclobutene carboxylic acid bridges with a low molecular weight compound having active amino groups in an aqueous buffer system, free of primary and secondary amines, at a pH of 7-10.
The selected compound contains preferably at least one amino group selected from a primary amino group and a secondary amino group.
The selected compound is expediently a polymer.
The polymer is advantageously a matrix.
The low-molecular weight compound contains at least one amino group selected from a primary amino group and a secondary amino group.
The present invention also relates to another method for manufacturing immobilized biomolecules and affinity ligands characterized by the steps of:
a) reacting dissolved polymer matrices having amino groups with a cyclobutene carboxylic acid derivative selected from the group consisting of cyclobutene carboxylic acid diester, cyclobutene carboxylic acid halide, cyclobutene carboxylic acid ester halide, cyclobutene carboxylic acid dialkoxy ester, and cyclobutene carboxylic acid imidazole as an activating compound in an inert solvent to form active matrices with active groups;
b) dissolving a compound selected from biomolecules, containing at least one amino group selected from a primary amino group and a secondary amino group, and affinity ligands, containing at least one amino group selected from a primary amino group and a secondary amino group;
c) incubating the activated matrices with the dissolved compound of step b) at a pH of 7-10 and a temperature of +4xc2x0 C. to +60xc2x0 C. in an aqueous buffer system, free of primary and secondary amines; and
d) reacting the incubated matrices of step c) with a low molecular weight compound having active amino groups in an aqueous buffer system, free of primary and secondary amines, at a pH of 7-10.
The low-molecular weight compound contains at least one amino group selected from a primary amino group and a secondary amino group.
The present invention further relates to a compound of the formula 
wherein
R1 and R2 are identical or different and are
hydrogen, cycloalkyl with 3 to 6 carbon atoms, linear or branched alkyl with up to 8 carbon atoms,
substituted with halogen or linear or branched alkoxy with up to 6 carbon atoms or hydroxy;
phenyl or benzyl,
substituted with halogen, nitro group, cyano group, carboxy, linear or branched alkyl, alkoxy, acyl or alkoxycarbonyl with up to 6 carbon atoms, respectively, or a group of the formula xe2x80x94NR3R4, wherein
R3 and R4 are identical or different and are hydrogen or linear or branched alkyl with up to 6 carbon atoms,
A is oxygen or sulfur,
B is oxygen or sulfur,
PL is, as a function of the meaning of PM, a compound functionalized with amino groups,
PM is a watersoluble or waterinsoluble matrix of natural or synthetic origin with covalently bonded primary or secondary amino groups, cells, cell organelles, or tissue samples.
Preferably, PL is an enzyme, Protein A, Protein B, Protein G, immunoglobulin or their fragments or aminocyclodextrin.
The matrix preferably consists of cellulose, polystyrene, polypropylene, polycarbonate, or glass. The invention relates to compounds of the general formula (I) 
wherein
R1 and R2 are identical or different and are: hydrogen, cycloalkyl with 3 to 6 carbon atoms or linear or branched alkyl with up to 8 carbon atoms, optionally substituted with halogen or linear or branched alkoxy with up to 6 carbon atoms, or hydroxy, phenyl or benzyl, optionally substituted with halogen, a nitro group, cyano group, carboxy group, linear or branched alkyl, alkoxy, acyl, or alkoxycarbonyl with respectively up to 6 carbon atoms, or a group of the formula xe2x80x94NR3R4, wherein
R3 and R4 are identical or different and are hydrogen or linear or branched alkyl with up to 6 carbon atoms,
A is oxygen or sulfur,
B is oxygen or sulfur,
PL
as a function of the respective meaning of PM
is a protein or
an enzyme, for example, alkaline phosphatase, xcex2-galactosidase, T4 PN kinase, horseradish peroxidase, phospholipase C or, for example, proteinase such as papain, pepsin, proteinase K, carboxypeptidase, endoproteinase Arg-C, bromelain, collagenase, dipeptidyl peptidase IV, or nuclease, for example, RNase A, RNase T1, RNase T2, DNase I, Bal 31, or oxidase, for example, alcohol oxidase, glucose oxidase, or esterase, for example, phosphodiesterase I and II, or glucosidase, for example, xcex2-glucoronidase, heparinas I, or lectin, for example, concanavalin A, or interleukin,
or
for a protein A
or protein G
or immunoglobulin of the type IgM, IgG, IgE, IgA and their fragments
or other compounds functionalized with amino groups, for example, biochemical probes, physiological indicators, chelates, lipids, tracers, oligopeptides, oligonucleotides, oligosaccharides, respectively, the corresponding monomer components or antibiotics,
PM
is a watersoluble or waterinsoluble matrix or natural or synthetic origin with covalently bonded primary or secondary amino groups in different embodiments (test tubes, microtiter plates, microscope slides, beads, membranes, resins, filters), which matrix, for example, is comprised of cellulose, polystyrene, polypropylene, polycarbonate, polymethacrylate, polyamide, or glass,
or different cells, cell organelles, artificial liposomes, and tissue samples,
or may have the meaning of PL.
The invention relates to preferably to compounds of the general formula (I) wherein
R1 and R2 are identical or different and are: hydrogen, cycloalkyl with 3 to 4 carbon atoms or linear or branched alkyl with up to 6 carbon atoms, optionally substituted with halogen or linear or branched alkoxy with up to 4 carbon atoms, or hydroxy, phenyl or benzyl, optionally substituted with halogen, a nitro group, cyano group, carboxy group, linear or branched alkyl, alkoxy, acyl, or alkoxycarbonyl with respectively up to 4 carbon atoms, or a group of the formula xe2x80x94NR3R4, wherein
R3 and R4 are identical or different and are hydrogen or linear or branched alkyl with up to 4 carbon atoms,
A is oxygen or sulfur
B is oxygen or sulfur,
PL
as a function of the respective meaning of PM
is a protein or
enzyme such as, for example, alkaline phosphatase, xcex2-galactosidase, T4 PN kinase, horseradish peroxidase, phospholipase C or, for example, proteinase such as papain, pepsin, proteinase K, carboxypeptidase, endoproteinase Arg-C, bromelain, collagenase, dipeptidyl peptidase IV, or nuclease such as, for example, RNase A, RNase T1, RNase T2, DNase I, Bal 31, or oxidase such as, for example, alcohol oxidase, glucose oxidase, or esterase such as, for example, phosphodiesterase I and II, or glucosidase such as, for example, xcex2-glucoronidase, heparinas I, or lectin such as, for example, concanavalin A or interleukin
or a protein A
or a protein G,
or immunoglobulin of the type IgM, IgG, IgE, IgA and their fragments,
or other compounds functionalized with amino groups as, for example, biochemical probes, physiological indicators, chelates, tracers, oligopeptides, oligonucleotides, oligosaccharides, respectively, the corresponding monomer components, or antibiotics,
PM
is a watersoluble or waterinsoluble matrix in various embodiments (test tubes, microtiter plates, microscope slides, beads, membranes, resins, filters) of natural or synthetic origin with covalently bonded primary or secondary amino groups, which matrix is, for example, comprised of cellulose, polystyrene, polypropylene, polycarbonate, polymethacrylate, polyamide, or glass,
or also different cells, cell organelles, artificial liposomes, and tissue samples,
or may also have the meaning of PL.
The invention relates especially to compounds of the general formula (I) wherein
R1 and R2 are identical or different and are: hydrogen, linear or branched alkyl with up to four carbon atoms, optionally substituted by halogen, or linear or branched alkoxy with up to 3 carbon atoms, or hydroxy, is benzyl, which is optionally substituted with halogen, a nitro group, cyano group, carboxy group, linear or branched alkyl, alkoxy, acyl, or alkoxycarbonyl with respectively up to 4 carbon atoms, or a group of the formula xe2x80x94NR3R4, wherein
R3 and R4 are identical or different and are hydrogen or linear or branched alkyl with up to four carbon atoms,
A, B is oxygen
PL
as a function of the respective meaning of PM
is a protein
or an enzyme, as, for example, alkaline phosphatase, xcex2-galactosidase, T4 PN kinase, horseradish peroxidase, phospholipase C or, for example, proteinase such as papain, pepsin, proteinase K, carboxypeptidase, endoproteinase Arg-C, bromelain, collagenase, dipeptidyl peptidase IV, or nuclease such as, for example, RNase A, RNase T1, RNase T2, DNase I, Bal 31, or oxidase such as, for example, alcohol oxidase, glucose oxidase, or esterase such as, for example, phosphodiesterase I and II, or glucosidase such as, for example, xcex2-glucoronidase, heparinas I, or lectin such as, for example, concanavalin A or interleukin
or Protein A,
or Protein G,
or biochemical probes, physiological indicators, chelates, tracers, oligopeptides, oligonucleotides, oligosacharides, respectively, the respective monomer components, or antibiotics, or immunoglobulin of the Type IgM, IgG, IgE, IgA and their fragments,
a watersoluble or waterinsoluble matrix of natural or synthetic origin in various embodiments (test tubes, microtiter plates, microscope slides, beads, membranes, resins, filters) with covalently bonded primary or secondary amino groups, which matrix is, for example, comprised of cellulose, polystyrene, polypropylene, polycarbonate, polymethacrylate, polyamide, or glass,
or various cells, cell organelles, artificial liposomes, and tissue
samples,
or may also have the meaning of PL.
The invention relates to immobilized biomolecules and affinity ligands characterized in that the immobilization reagents are cyclobutene carboxylic acid derivatives that bond the covalent biomolecules and affinity ligands to a matrix.
The invention also relates to immobilized biomolecules and affinity ligands characterized in that the immobilization reagent is a cyclobutene carboxylic acid derivative (I) having the general chemical structural formula as follows: 
wherein
R1 and R2 are identical or different and are:
hydrogen, cycloalkyl with 3 to 6 carbon atoms or linear or branched alkyl with up to 8 carbon atoms, optionally substituted with halogen, or linear or branched alkoxy with up to 6 carbon atoms or hydroxy, phenyl or benzyl, optionally substituted with halogen, nitro group, cyano group, carboxy group, linear or branched alkyl, alkoxy, acyl, or alkoxycarbonyl with respectively up to 6 carbon atoms, or a group of the formula xe2x80x94NR3R4,
wherein R3 and R4 are identical or different and are hydrogen or linear or branched alkyl with up to 6 carbon atoms,
A is oxygen or sulfur,
B is oxygen or sulfur,
PL, as a function of the respective meaning of PM, is a protein or an enzyme, such as, for example, alkaline phosphatase, xcex2-galactosidase, T4
PN kinase, horseradish peroxidase, phospholipase C or, for example, proteinase such as papain, pepsin, proteinase K, carboxypeptidase, endoproteinase Arg-C, bromelain, collagenase, dipeptidyl peptidase IV, or nuclease, such as, for example, RNase A, RNase Ti, RNase T2, DNase I, Bal 31, or oxidase, such as, for example, alcohol oxidase, glucose oxidase, or esterase such as, for example, phosphodiesterase I and II, or glucosidase, such as, forexample, xcex2-glucuronidase, heparinase I, or lectin, such as, for example, concanavalin A, or interleukin,
or
a protein A or protein G
or
an immunoglobulin of the type IgM, IgG, IgE, IgA and their fragments
or
compounds functionalized with amino groups such as, for example, biochemical probes, physiological indicators, chelates, tracers, oligopeptides, oligonucleotides, oligosaccharides, respectively, the corresponding monomeric components, or antibiotics,
PM is a watersoluble or waterinsoluble matrix of a natural or synthetic origin in various embodiments (test tubes, microtiter plates, microscope slides, beads, membranes, resins, filters) with covalently bonded primary or secondary amino groups, such as, for example cellulose, polystyrene, polypropylene, polycarbonate, polymethacrylate, polyamide, or glass,
or
different cells, cell organelles, artificial liposomes, and tissue samples or may have the meaning of PL.
Preferably, the biomolecules and affinity ligands which must contain at least one primary or secondary amino group, are activated with a cyclobutene carboxylic acid diester or cyclobutene carboxylic acid halide to form a cyclobutene carboxylic acid adduct and are subsequently reacted with any suitable matrix which must contain primary or secondary amino groups, under formation of covalent cyclobutene carboxylic acid bridges.
Biotin derivatives such as preferably biocytin are reacted according to the inventive method with cyclobutene carboxylic acid diethyl esters in ethanol or dimethylformamide in the presence of triethyl amine or pyridine as a base at room temperature to the adduct AL1 
and subsequently are reacted preferably with polymethacrylate with aliphatic amino groups (particle size: 65 xcexc, 100 xcexcmol/ml amine, AF-amino-650 M, Tosohaas in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALa as a compound of the general formula (I): R1, R2=H; A,B=O; PM=polymethacrylate; PL=bicytinyl.
Antibiotics, preferably daunomycin, are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester and ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to the adduct AL2 and subsequently are reacted, preferably with polymethacrylate, functionalized with aliphatic amino groups (particle size: 65 xcexc, 100 xcexcmmol/ml amine, AF-amino-650 M, Tosohaas) in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized antibiotic ALb as a compound of the general formula (I): R1, R2=H; A, B=O; PM=polymethacrylate; PL=daunomycinyl 
Oligopeptides, as preferably glutathione, are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to the adduct AL3 
and subsequently reacted with polymethacrylate functionalized with aliphatic amino groups (particle size: 6xcexc, 100 xcexcmol/ml amine, AF-amine-650 M, Tosohaas) in ethanol or dimethylformamide in the presence of triethyl amine or pyridine as a base or also in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALc as a compound of the general formula (I): R1, R2=H; A, B =O; PM=polymethacrylate; PL=gluthationyl.
Penicillin compounds, preferably amoxicillin, is reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to the adduct AL4 and 
subsequently preferably is reacted with polymethacrylate, functionalized with aliphatic amino groups (particle size 65xcexc, 100 xcexcmol/ml amine, AF-amino-650 M, Tosohaas) in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or also in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALd as a compound of the general formula (I): R1, R2=H; A, B=O; PM=polymethacrylate; PL=amoxicillyl.
Antimycotics such as preferably amphotericin B are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to form the adduct AL5 and subsequently reacted preferably polyamides functionalized with amino groups (preferably Biodyne A, Pall) in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALe as a compound of the general formula (I): R1, R2=H; A, B=O; PM=polyamide; PL=amphotericinyl B. 
Chelate forming compounds such as preferably iron ion-selective desferrioxamine B are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to form the adduct AL6 and subsequently reacted preferably with polyamides, which are functionalized with aliphatic amino groups, in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALf as a compound of the general formula (I): R1, R2=H; A, B=O; PM=polyamide; PL=desferrioxaminyl B. 
Amino-functionalized crown ethers such as preferably 2-aminomethyl-12-crown-4 are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to form the adduct AL7 and subsequently reacted preferably with a polyamide membrane (Byodyne A, Pall GmbH) in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligands ALg as a compound of the general formula (I): R1, R2=H; A, B=0; PM=polyamide; PL=2-aminomethyl-12-crown-4. 
Chelate forming compounds such as preferably substituted DOTA are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to form the adduct AL8 and subsequently reacted with a polyamide membrane functionalized with amino groups (Biodyne A, Pall) in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer system (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALh as a compound of the general formula (I): R1, R2=H; A, B=O; PM=polyamide; PL=aminobutyl-DATA. 
Amino sugars such as preferably aminocyclodextrin are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to the adduct AL9 and subsequently reacted with polyacrylate functionalized with aliphatic amino groups (particle size: 65xcexc, 100 xcexcmol/ml amine; AF-amino-650 M, Tosohaas) in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALi as a compound of the general formula (I): R1, R2=H; A, B=O; PM=polymethacrylate; PL=aminocyclodextrinyl. 
Amino substituted DNA components such as 3-aminopropyl-adenine are reacted according to the present invention with cyclobutene carboxylic diethyl ester and ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to form the adduct AL10 and subsequently reacted preferably with glass beads which are functionalized with amino groups (aminopropyl glass, Sigma) in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALj as a compound of the general formula (I): R1, R2=H; A, B=O; PM=glass beads; PL=3-propyl-(-3-yl)-adenine. 
Dyes or indicators such as preferably amino-substituted fluorescein derivatives are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to form the adduct AL11 and subsequently reacted preferably with glass beads functionalized with aliphatic amino groups in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligands ALk as a compound of the general formula (I): R1, R2=H; A, B=O; PM glass beads; PL=5-((5-pentenyl)-thioureidyl)fluorescein. 
Steroids such as, for example, aminodigitoxigenin are reacted according to the present invention with cyclobutene carboxylic acid diethyl ester in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature to form the adduct AL12 and subsequently reacted with oligonucleotides functionalized with amino groups such as preferably 5xe2x80x2-amino-polydG in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALh as a compound of the general formula (I): R1, R2=H; A, B=O; PM=5xe2x80x2-yl-polydG; PL=digitoxigenyl. 
Amino-functionalized oligonucleotides such as preferably 5xe2x80x2-Amino-poly(dG)12 are reacted according to the present invention in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base at room temperature or in aqueous buffer systems of pH 7.5 with cyclobutene carboxylic acid diethyl ester to form the adduct AL13 and subsequently reacted preferably with polyamide membranes functionalized with aliphatic amino groups (Biodyn A, Pall) in ethanol or dimethylformamide in the presence of triethylamine or pyridine as a base or in aqueous buffer systems (carbonate, Borate) at pH 9.0 at room temperature to form the immobilized affinity ligand ALm as a compound of the general formula (I): R1, R2=H; A, B=O; PM=polyamide; PL=5xe2x80x2-yl-poly(dG)12. 
Polymer matrices or affinity ligands, which contain primary or secondary amino groups, are reacted according to the present invention with cyclobutene carboxylic acid diester, cyclobutene carboxylic acid halide, or cyclobutene carboxylic acid ester halide as activating reagents under formation of active groups to activated matrices or activated affinity ligands.
Biomolecules and affinity ligands PL and/or PM, which must contain at least one primary or secondary amino group, are reacted according to the present invention with a selected activated matrix under formation of a covalent bond or a plurality of stable covalent bonds.
A polymer matrix or affinity ligands, which contain primary or secondary amino groups, are reacted according to the present invention with cyclobutene carboxylic acid diesters, cyclobutene carboxylic acid halides, or cyclobutene carboxylic acid ester halide at 0C. to +100xc2x0 C., preferably at +100 C. to +60xc2x0 C.
The reaction is preferably carried out in a conventional organic solvent, that will not change during reaction, with less than molar or greater than molar amounts with respect to the amino groups.
The activation is carried out in the presence of organic bases, preferably triethylamine or pyridine, and these bases are added in an amount of 1 mol to 100 mol, preferably 1 mol to 10 mol, relative to 1 mol of the compound of the general formula (I).
The reaction time varies as a function of the selected reaction components and the reaction temperature, whereby times of 0.1 to 10 hours are preferred.
As a polymer matrix or as an affinity ligand all synthetic as well as natural compounds are suitable which contain covalent primary or secondary amino groups.
As a reaction medium all aqueous buffer system are suitable such as preferably phosphate, citrate, borate, carbonate, or mixtures thereof.
The concentration of the molecules to be conjugated can vary in wide ranges and is preferably for proteins to be coupled between 0.01 and 50 mg/ml.
The reaction can be performed in a temperature range of +4xc2x0 C. to +60xc2x0 C.
The pH value during the coupling reaction may be between 7 and 10.
The reaction time varies as a function of the selected reaction components, the reaction temperature, and the pH value and is normally between 0.1 and 50 hours.
The remaining active cyclobutene carboxylic acid groups after immobilization are deactivated according to the present invention with low molecular weight compounds that must contain at least one primary or secondary amino group.
As a blocking medium all aqueous buffer systems are suitable.
The concentration of the reactive groups for blocking can vary within wide ranges and is preferably for amino acids within a range between 0.01 and 100 mg/ml.
The reaction temperature for blocking can be within a temperature range of +4xc2x0 C. to +60xc2x0 C.
The pH value during the blocking reaction is between pH 7 and 10.
The reaction time as a function of the selected blocking compounds, the reaction temperature and the pH value is preferably between 0.1 and 50 hours.